Document e5N3EKLXy3j5mzG5L97O3Oy64

DEC-24-96 T'u'E 33:03 PM Huntingdon Life Sciences, Inc. P.Q. Box 2360, Mefflers Rood East MSstone, NJ 08875-2360 Teteptwoe: (908) *73-2000, L 7000 FaeamJa; (908) 373-3992.3899 Fax To: Robert A. Venezia, DrPH Pram: Dr, Michael J. Butler Fax: (703) 741-6091 Phone: (703)741-5639 Pages: 19 Data December 24,1996 Re: vinyl chlonde satellite Protocol, Version 2 CC: Ray Schroeder Urgant For Review G Please Comment G Please Reply Please Recycle e Comments: Dear Sob, Please find attached version 2 of the protocol for the referenced studies. This protocol was used to determine the prices sent over by Mike Butler earlier today. As always, please give me a call at (908) 873-2550, extension 2910, if I can assist further. Sincerely, Ray E. SetStudy Director CMA 118655 Huntingdon 2 January 1997 Robert A. Venezia, Ph D Chemical Manufacturers Association Chemstar Department 1300 Wilson Blvd. Arlington, VA 22209 RE: Studies to Evaluate the Formation and Repair of DNA Adducts Induced in Adult and Neonatal CD Rats by Vinyl Chloride (Study No 96-4092) Dear Dr. Venezia: Attached please find the protocol for the above referenced study A copy of this protocol was faxed to you on Tuesday, 24 December 1996. Best regards, Study Director RES/jb Attachment Huntingdon Life Sciences Inc. PO Box 2360, Menlers Road, East Millstone, NJ 08875-2360 USA. Tel: +1 908 873 2550 Fax: +1 908 873 3992 CMA 118656 Huntingdon HUNTINGDON LIFE SCIENCES STUDIES TO EVALUATE THE FORMATION AND REPAIR OF DNA ADDUCTS INDUCED IN ADULT AND NEONATAL CD RATS BY VINYL CHLORIDE STUDY NO.: ISSUE NO.: 96-4092 2 SUBMITTED TO: Chemical Manufacturers Association Chemstar Department ATTENTION: Robert A. Venezia, Ph.D. DATE: 24 December 1996 Huntingdon Life Sciences Inc. PO Box 2360, Mettlers Road, East Millstone, NJ 08873-2360 USA. Tel: +1 908 873 2350 Fax: +1 908 873 3992 CMA 118657 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 2 Protocol 02 1 INTRODUCTION: 1.1 STUDY NO.: 96-4092 1.2 ISSUE NO.: 2 1.3 STUDY TITLE: Studies to Evaluate the Formation and Repair of DNA Adducts Induced in Adult and Neonatal CD Rats by Vinyl Chloride 1.4 TEST MATERIAL: Vinyl Chloride 1.5 SPONSOR: Chemical Manufacturers Association Chemstar Department 1 300 Wilson Boulevard Arlington, Virginia 22209 1.6 SPONSOR REPRESENTATIVE: Robert A. Venezia, Ph.D. Phone - 703-741-5639 Fax - 703-741-6091 1.7 TESTING FACILITY: Huntingdon Life Sciences Mettlers Road P.O. Box 2360 East Millstone, NJ 08875-2360 1.8 PURPOSE: Several studies involving adult or neonatal animals will be performed to evaluate the formation and repair of DNA adducts induced by vinyl chloride. These studies will provide an improved understanding of the mechanisms of vinyl chloride carcinogenesis. These studies will also provide information relevant to cancer risk assessment of vinyl chloride. CMA 118658 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 3 Protocol 02 2 STUDY PERSONNEL: Study Director: Raymond E. Schroeder, M.S., DABT Alternate: Dean E. Rodwell, M.S. Vice-President of Toxicology Director of Toxicology: Carol S. Auletta, B.A., DABT Inhalation Toxicologist: Gary M. Hoffman, B.A., DABT Supervisor Rodent Toxicology: Ellen Whiting, AALAS LAT Primary Technician(s): Vice President, Pathology: Ward R. Richter, D.V.M., M.S., ACVP Additional personnel will be documented in the project file and presented in the final report. 3 REGULATORY REFERENCES: 3.1 TEST GUIDELINE: These studies are not designed to meet or exceed any particular guidelines. 3.2 GOOD LABORATORY PRACTICES: These studies will not be conducted in compliance with GLP guidelines. 3.3 FACILITIES MANAGEMENT/ANIMAL HUSBANDRY: Currently acceptable practices of good animal husbandry will be followed, e.g., Guide for the Care and Use of Laboratory Animals; DHHS Publication No. (NIH) 86-23, Revised 1985. Huntingdon Life Sciences (East Millstone, NJ) is fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). CMA 11865Q Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 4 Protocol 02 3.4 ANIMAL WELFARE ACT COMPLIANCE: This study will comply with all appropriate parts of the Animal Welfare Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 1 68, August 31,1989, pp. 361 1 2-361 63 effective October 30, 1 989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15,1991, pp. 6426-6505 effective March 18, 1991. The Sponsor should make particular note of the following: 1. The Sponsor's signature on this protocol documents for the study described, there are no generally accepted non-animal alternatives and the study does not unnecessarily duplicate previous experiments. 2. All procedures used in this study have been designed to avoid discomfort, distress and pain to the animals. All methods are described in this study protocol or in written laboratory standard operating procedures. 3. Any aspects of this study which cause more than momentary or slight pain or distress to the animals will be performed with appropriate sedatives, analgesics or anesthetics unless the withholding of these agents is justified for scientific reasons, in writing by the Sponsor and the Study Director, in which case the procedure will continue for the minimum time necessary. 4. Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanatized as deemed appropriate by the Testing Facility's veterinary staff and the Study Director. The Sponsor will be advised by the Study Director of all circumstances which could lead to this action in as timely a manner as possible. 5. Methods of euthanasia used during this study are in conformance with the above referenced regulations. 4 QUALITY ASSURANCE MONITORING: The Huntingdon Life Sciences Quality Assurance Unit (East Millstone, NJ) will not monitor the facilities, equipment, personnel, methods, practices, records and controls used in this study to assure that they are in conformance with this protocol and company standard operating procedures. CMA118660 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 5 Protocol 02 5 ALTERATION OF DESIGN: Alterations of this protocol may be made as the study progresses. No changes in the protocol will be made without the consent of the Sponsor. In the event that the Sponsor authorizes a protocol change verbally, such changes will be honored by the Testing Facility and will be followed by a written verification. All protocol modifications will be signed by the Study Director and a Sponsor representative. Any modifications potentially affecting animal welfare will also be signed by two members of the Institutional Animal Care and Use Committee prior to the modification's implementation. 6 PROPOSED STUDY DATES: Study No. 1 - Adult Rats Exposed to Vinyl Chloride for 1 Week Receipt of test animals: Initiation of exposures: Termination of exposures: * (to be addressed by amendment) * * Necropsy * Study No. 2 - Weanling Rats Exposed to Vinyl Chloride for 1 Week Initiation of exposures: * Termination of exposures: * Necropsy * Study No. 3 - Adult Rats Exposed to Vinyl Chloride for 4 Weeks Initiation of exposures: * Termination of exposures: * Necropsy * Study No. 4 - Adult Rats Exposed to [13C2l-Vinyl Chloride for 1 Week Initiation of exposures: Termination of exposures: * * Necropsy * CMA 118661 Huntingdon Life Sciences Study No. 96*4092 Chemical Manufacturers Assoc. 7 EXPERIMENTAL DESIGN: Page 6 Protocol 02 Study type 1 - Adult Male Rats 2 - Wean ling Male Rats 3 - Adult Male Rats 4 - Adult Male Rats Exposure Levels (ppm) 0. 10, 100, 1 100b 0, 10, 100, 1 100b No. of Animals8 32 (8/group) 32 (8/group) T reatment Schedule 5 days, 6 hrs/day 5 days, 6 hrs/day 0, 10, 100, 1 100b 64 (1 S/group) 5 days/week, four weeks, 6 hrs/day 10, 100, 1 100c 24 (8/group) 5 days, 6 hrs/day Necropsy Schedule All animals following exposure Day 5 All animals fallowing exposure Day 5 8 animals/group following last exposure (Week 4) and 8 animals/group following one week recovery All animals following exposure Day 5 8 1 -2 "extra" animals will be exposed 6 hrs/day at each dose level in each of these studies to provide whole tissues at terminal sacrifice. No additional data would be collected for these animals and they would not be perfused b Whole body exposures conducted during the performance of a vinyl chloride two-generation reproduction study (Huntingdon Life Sciences Study No. 96-4080), c Nose-only exposure with (13C2) - vinyl chloride. 8 TEST MATERIAL: 8.1 TEST MATERIAL (Studies 1, 2 and 3): Vinyl Chloride Description, lot number, storage, expiration date and handling procedures, as well as other pertinent information will be documented in the study data. Properties of the test material are presented in the Appendix B. 8.2 IDENTIFICATION OF TEST MATERIAL (Studies 1, 2 and 3): Unless otherwise noted, the identity, strength, purity, composition, stability, and method of synthesis, fabrication and/or derivation of each batch of the test material will be documented by the Sponsor before its use in the study. 8.3 ANALYSIS OF THE TEST MATERIAL (Studies 1,2 and 3): The purity of the lot of test material used on study will be the determined from periodic assessments of the test material over the course of the study. These analyses will be performed at the Testing Facility. CMA 118662 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 7 Protocol 02 8.4 TEST MATERIAL (Study No.4): [13C2]-Vinyl Chloride Test material for this study will be supplied by Cambridge Isotope Laboratoies. The Sponsor or the Supplier will be responsible for the characterization. Unless otherwise noted, the identity, strength, purity, composition, stability, and method of synthesis, fabrication and/or derivation for the batch of test material used in Study 4 will be documented by the Sponsor before its use. 9 TEST ANIMALS: Albino Rats (Outbred) VAF/Plus9 9.1 STUDY NO. 1, ADULT RATS EXPOSED TO VINYL CHLORIDE FOR 1 WEEK: 9.1.1 Species/Strain: Albino Rats/Sprague Dawley - derived (CD'9) [Crl: CD9 BR] 9.1.2 Supplier: Charles River Laboratories Portage, Michigan 9.1.3 Animal requirements/specifications: 9.1.3.1 Number: Eight per group for a total of 32 animals on study. Approximately 40 animals will be purchased and acclimated. The unselected study animals will be designated "extra" animals and be distributed equally among the four study groups. These "extra" animals will be exposed the same as study animals but will be used only to provide tis sues/organs at terminal sacrifice. No other data will be collected for these animals. 9.1.3.2 Sex and Age: Males approximately 11 weeks at receipt and 12 weeks at initiation of treatment. 9.1.3.3 Acclimation period: At least one week; all animals will be observed twice daily. Prior to assignment to study all animals will be examined to determine suitability for use. CMA 118663 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 8 Protocol 02 9.2 STUDY NO. 2, WEANLING RATS EXPOSED TO VINYL CHLORIDE FOR 1 WEEK: 9.2.1 Species/Strain: Albino Rat/Sprague Dawley - derived (CD*) [Crl: CD* BR] 9.2.2 Supplied by: Unselected F1 pups from control litters produced during the twogeneration reproduction study (Study No. 96-4080). 9.2.3 Animal requirements/specifications: 9.2.3.1 Number: Eight per group for a total of 32 animals on study. Approximately 40 Ft pups will be identified for use in this study. The unselected study animals will be designated "extra" animals and be distributed equally among the four study groups. These "extra" animals will be exposed the same as study animals but will be used only to provide tissues/ organs at terminal sacrifice. No other data will be collected for these animals. 9.2.3.2 Sex and Age: Postnatal Day 26 weanling male pups (weaned on Postnatal Day 25). 9.2.3.3 Acclimation: None - prior to assignment to study all pups will be examined to determine suitability for use. 9.3 STUDY NO. 3, ADULT RATS EXPOSED TO VINYL CHLORIDE FOR 4 WEEKS 9.3.1 Species/Strain: Albino Rat/Sprague Dawley - derived (CD*) [Crl: CD BR] 9.3.2 Supplier: Charles River Laboratories Portage, Michigan CMA 118664 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 9 Protocol 02 9.3.3 Animal requirements/specifications: 9.3.3.1 Number: Sixteen animals per group for a total of 64 animals on study. More animals then required for study will be purchased and acclimated. Approximately 80 animals will be identified for use in this study. The unselected study animals (about 16 animals total) will be designated "extra" animals and be distributed equally among the four study groups. These "extra" animals will be exposed the same as study animals but will be used only to provide tissues/organs at terminal sacrifice. No other data will be collected for these animals. 9.3.3.2 Sex and Age: Males approximately 1 1 weeks at receipt and 1 2 weeks at initiation of treatment. 9.3.3.3 Acclimation: At least one week; all animals will be observed twice daily. Prior to assignment to study all animals will be examined to determine suitability for use. 9.4 STUDY 4, ADULT RATS EXPOSED TO [13C2] - VINYL CHLORIDE FOR 1 WEEK: 9.4.1 Species/Strain: Albino Rat/Sprague Dawley - derived (CD'9) [Crl: CD BR] 9.4.2 Supplier: Charles River Laboratories Portage, Michigan 9.4.3 Animal requirements/specifications: 9.4.3.1 Number: Eight animals per group for a total of 24 animals on study. More animals then required for study will be purchased and acclimated. Approximately 30 animals will be identified for use in this study. The unselected study animals (about 6 animals total) will be designated "extra" animals and be distributed equally among the four study CMA118665 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 10 Protocol 02 9.4.3.1 Number: groups. These "extra" animals will be exposed the same as study animals but will be used only to provide tissues/organs at terminal sacrifice. No other data will be collected for these animals. 9.4.3.2 Sex and Age: Males approximately 11 weeks at receipt and 1 2 weeks at initiation of treatment. 9.4.3.3 Acclimation: At least one week; all animals will be observed twice daily. Prior to assignment to study all animals will be examined to determine suitability for use. 9.5 ANIMAL HUSBANDRY 9.5.1 Housing: Animals will be housed in suspended, stainless steel cages with wire mesh fronts and floors. During non-exposure periods when urine is to be collected, animals will be housed individually in stainless steel, suspended cages designed to collect urine. 9.5.2 Food: Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO). Each animal's cage will be fitted to retain a glass feeder cup with a stainless steel lid. Feed will be available ad libitum during non exposure periods. 9.5.3 Water: Facility water supply (Elizabethtown Water Company, Westfield, NJ); without restriction during exposures and non-exposure periods via an automated water delivery system to individual animal cages in Studies 1-3. Animals in Study No. 4 will not have access to water during the exposure period. 9.5.4 Feed Analysis: Analytical certification of batches of feed used during the study which are provided by the manufacturer, will be maintained on file at the Testing Facility. There are no known contaminants in the feed which are expected to interfere with the results of this study. CMA118666 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 11 Protocol 02 9.5.5 Water Analysis: Monthly water analyses, provided by the supplier, will be maintained on file at the Testing Facility. Biannual chemical and microbiological analyses of water samples collected from representative rooms in this facility will be conducted to assure that water being provided meets standards specified under the EPA National Primary Drinking Water Regulations (40 CFR Part 141). Results will be maintained on file. There are no known contaminants in the water which are expected to interfere with the results of this study. 9.5.6 Veterinary Care: Animals will be monitored by the technical staff for any conditions requiring possible veterinary care. If any such conditions are identi fied, a staff veterinarian will be notified for an examination and evalua tion. Any medical veterinary intervention will be made only with approval of the staff veterinarian and the Study Director. The Sponsor will be consulted whenever possible. However, in emergency situations, decisions will be made as needed and the Sponsor will be advised as soon as possible. 9.5.7 Environmental Conditions: 9.5.7.1 Light/Dark Cycle: Twelve hour light/dark cycle daily. 9.5.7.2 Temperature: Monitored and recorded twice daily. Temperature in the animal room and exposure chambers will be maintained in the range of approxi mately 20-24C to the maximum extent possible. 9.S.7.3 Humidity: Humidity in the animal quarters will be monitored and recorded once daily. The desired humidity range in the animal quarters is 40-70% and 40-60% in the chambers during exposures. Humidity will be maintained in this range to the maximum extent possible. CMA 118667 Huntingdon Life Sciences Study No, 96-4092 Chemical Manufacturers Assoc. Page 12 Protocol 02 9.6 SELECTION FOR STUDY: In Studies No. 1,3 and 4 animals will be sorted into groups so as to equalize the mean body weights between groups. A manual sorting procedure will be used. In Study No. 2 animals will be manually sorted among the groups to equalize as best possible the distribution of pups from each F1 litter. 9.7 ANIMAL IDENTIFICATION: Each animal will be assigned a temporary identification number upon receipt. After selection for study, each animal will be ear-tagged (metal eartag) with a number assigned by the Testing Facility, This number plus the study number will comprise the unique identification for each study animal. 10 TEST MATERIAL ADMINISTRATION: 10.1 ROUTE OF ADMINISTRATION: Studies No. 1, 2 and 3 - whole body inhalation; Study No. 4 - nose only inhalation. 10.2 JUSTIFICATION FOR ROUTE OF ADMINISTRATION: The inhalation route is one of the potential routes of human exposure to this test material. 10.3 FREQUENCY OF ADMINISTRATION: 10.3.1 Study No. 1: Five consecutive days, 6 hrs/day. 10.3.2 Study No. 2: Five consecutive days, 6 hrs/day. 10.3.3 Study No. 3: Five consecutive days per week, 6 hrs/day, for four weeks. 10.3.4 Study No. 4: Five consecutive days per week, 6 hrs/day. CMA 118668 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc, Page 13 Protocol 02 10.4 INHALATION EXPOSURE GENERATION PROCEDURE: 10.4.1 Studies 1, 2 and 3: The test material will be administered as a gas in the breathing air of the animals. The test atmospheres will be generated by an appropriate procedure to be developed at the Testing Facility. The generation method will be described in the final report and maintained with the data package for this study. The whole-body exposure chambers will have a volume of approxi mately 6000 liters (6 m3). Each chamber will be operated at a minimum flow rate of 1 200 liters per minute. The final airflow will be set to provide at least one air change in 5.0 minutes (12 air chang es/hour) and a T99 equilibrium time of approximately 23 minutes. This chamber size and airflow rate are considered adequate to maintain the oxygen level above 19% and the animal loading factor below 5%. 10.4.2 Study No. 4: Dynamic nose-only exposure operating at approximately 2.4 Ipm of oxygen enriched air (40%). 10.5 CHAMBER MONITORING: A nominal exposure concentration will be calculated, if possible. The flow of air through the chamber/nose only generation apparatus will be monitored using appropriate calibrated equipment. The test material consumed during the exposure will be divided by the total volume of air passing through the chamber (volumetric flow rate times total exposure time) to give the nominal concentration. During each test material exposure, measurements of airborne concentrations will be made at least hourly using a MIRAN infrared spectrophotometer. Also prior to initiating of animal exposures, additional samples will be taken to determine the distribution of the test material in the exposure chamber. During each week of exposure, particle size determinations will be performed using an appropriate instrument to characterize the aerodynamic particle size distribution of any aerosol present. CMA 118669 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 14 Protocol 02 10.5 CHAMBER MONITORING: The minimum frequency of chamber activity is summarized below: Activity Analytical Test Material Concentration Temperature Relative Humidity Airflow Rate Nominal Test Material Concentration (excluding the control chamber) Rotation Pattern of Exposure Cages Loading/Unloading Verification Frequency/chamber/day 6 times (hourly) 7 times 7 times 7 times once once once 10.6 EXPOSURE CONCENTRATIONS: For all studies the targeted exposure concentrations of vinyl chloride will be 0,10,100 and 1100 ppm. These exposure levels correspond to oral equiva lent doses of approximately 0, 9.2, 92 and 1012 mg/kg/day assuming ventilation rates of 1 L/min/kg, 100 percent absorption and a 6 hr/day exposure. The high concentration of vinyl chloride was selected based on the oral equivalent of the limit dose of 1000 mg/kg body weight/day. The high exposure level of 1100 ppm is also expected to produce effects on the liver and other organ systems (ATSDR, 1993). The middle and low exposure levels were selected to provide a dose response for the observed effects and a no-observed-effect level, respectively. C/WA 118670 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. 11 EXPERIMENTAL EVALUATIONS: Page 1 5 Protocol 02 11.1 OBSERVATIONS: 11.1.1 Viability Determinations: Observations for mortality, general appearance and signs of severe toxic or pharmacologic effects as well as availability of feed and water will be made at least twice daily (morning and afternoon). Animals in extremely poor health or in a possible moribund condition will be identified for further monitoring and possible euthanasia. All animals found dead will be submitted for a gross macroscopic examination. Animals found dead after normal working hours will be refrigerated until a necropsy can be performed. 11.2 BODY WEIGHTS: 11.2.1 Study No. 1: Animals will be weighed pretest, on the day treatment initiates and at terminal sacrifice. The "extra" animals in each group will be weighed at study start and at terminal sacrifice. 11.2.2 Study No. 2: Animals will be weighed pretest (Day 25), on the day treatment initiates and at terminal sacrifice. The "extra" pups included on study will be weighed at study start and at terminal sacrifice. 11.2.3 Study No. 3: Animals will be weighed pretest, on the day treatment initiates, weekly thereafter and at terminal sacrifice. The "extra" pups included on study will be weighed at study start and at terminal sacrifice. 11.2.4 Study No. 4: Animals will be weighed pretest, on the day treatment initiates and at terminal sacrifice. The "extra" pups included on study will be weighed at study start and at terminal sacrifice. CMA 118671 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 16 Protocol 02 11.3 FOOD CONSUMPTION: Food consumption will not be recorded during these studies. 11.4 URINE COLLECTION: 11.4.1 Study No. 3: Urine will be collected over the non-exposure interval (a period of approximately 1 7 hrs) on Day 1,5,10 and 19 from all animals in the group exclusive of the "extra" animals in each group). Urine from each animal on each day will be collected on ice and stored refrigerat ed until being transferred to Dr. Swenberg's lab at the University of North Carolina for evaluation. 11.4.2 Study No. 4: Urine will be collected for each animal for three days prior to exposure and during the non-exposure period on Days 1-4. Urine from each animal on each day will be collected on ice and stored refrigerated until being transferred to Dr. Swenberg's lab (University of North Carolina) for evaluation. 12 POSTMORTEM: 12.1 SCHEDULED SACRIFICE: 12.1.1 Study No. 1: All eight rats per group and all "extra" dosed animals on Day 5 after the last exposure. 12.1.2 Study No. 2: All eight rats per group and all "extra" dosed animals on Day 5 after the last exposure. 12.1.3 Study No. 3: Eight rats per group and two "extra" dosed animals per group will be sacrificed on the last day of exposure (end of Week 4), The remaining animals in each group to include the remaining "extra" animals will be sacrificed seven days after the last exposure (following a one week period when animals will not have been exposed to vinyl chloride). CMA 118672 Huntingdon Life Sciences Study No. 96-4092 Chemical Manufacturers Assoc. Page 1 7 Protocol 02 12.1.4 Study No. 4: All 8 rats per group and the "extra" animals in each group will be sacrificed on Day 5 after the last exposure. 12.2 METHOD OF EUTHANASIA: Studies No. 1-4: Whole body perfusion following anesthesia with carbon dioxide. The "extra" animals will be sacrificed by exsanguination following anesthesia from inhaled carbon dioxide. 12.3 NECROPSY: Studies No. 1-4: The sacrifice, perfusion and necropsy of these animals will be performed under the supervision of a technical staff from Dr. James Swenberg's laboratory at University of North Carolina and all data generated during these examinations will be their responsibility and retained at his laboratory. The "extra" animals in each group within these studies will not be perfused but will be used to collect whole tissues at necropsy. The tissues will be transferred to Dr. Swenberg's laboratory at University of North Carolina. 13 PRESERVATION OF RECORDS AND SPECIMENS: All data documenting experimental details and study procedures and observa tions will be recorded and maintained as raw data. At the completion of the study, all reports, raw data, preserved specimens and retained samples will be maintained in the Testing Facility's Archives for a period of 10 years after submission of the signed final report. The Sponsor will be contacted in order to determine the final disposition of these materials. The Sponsor is responsible for all costs associated with the storage of these materials beyond 10 years from the issuance of the final report and for any costs associated with the shipment of these materials to the Sponsor or to any other facility designated by the Sponsor. 14 REPORT: The reports for each of these studies {No. 1-4) will be prepared by Dr. James Swenberg, University of North Carolina. Copies of all in-life data generated by Huntingdon Life Sciences will be sent to Dr. Swenberg, University of North Carolina, Dept, of Environmental Sciences and Engineering, Campus Box 7400, Rosenau Hall, Chapel Hill, NC 27599-7400. The original raw data will be retained in the study file. CMA 118673 Huntingdon Life Sciences Study No. 96*4092 Chemical Manufacturers Assoc. Page 18 Protocol 02 15 SIGNATURES: 15.1 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC): The IACUC Protocol Review Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations. BY: DATE: TITLE: FOR: Institutional Animal Care and Use Committee Member Huntingdon Life Sciences Institutional Animal Care and Use Committee BY: DATE: TITLE: FOR Institutional Animal Care and Use Committee Member Huntingdon Life Sciences Institutional Animal Care and Use Committee 15.2 PROTOCOL REVIEWED AND ACCEPTED: BY: DATE: TITLE: FOR: Raymond E, Schroeder, M.S., DABT Study Director Huntingdon Life Sciences BY: DATE: TITLE: FOR: Robert A. Venezia, Ph.D. Sponsor Representative Chemical Manufacturers Association Chemstar Department CMA 118674